Two-site microendoscopic imaging probe for simultaneous three-dimensional imaging at two anatomic locations in tissues.

Systems that can image in three dimensions at cellular resolution and across different locations within an organism may enable insights into complex biological processes, such as immune responses, for which a single location measurement may be insufficient. In this Letter, we describe an in vivo two-site imaging probe (TIP) that can simultaneously image two anatomic sites with a maximum separation of a few centimeters. The TIP consists of two identical bendable graded index (GRIN) lenses and is demonstrated by a two-photon two-color fluorescence imaging system. Each GRIN lens has a field of view of 162 × 162 × 170 µm3, a nominal numerical aperture of 0.5, a magnification of 0.7, and working distances of 0.2 mm in air for both ends. A blind linear unmixing algorithm is applied to suppress bleedthrough between channels. We use this system to successfully demonstrate two-site two-photon two-color imaging of two biomedically relevant samples, i.e., (1) a mixture of two autofluorescent anti-cancer drugs and (2) a live hybrid tumor consisting of two spectrally distinct fluorescent cell lines.

In vivo visualization of butterfly scale cell morphogenesis in Vanessa cardui.

During metamorphosis, the wings of a butterfly sprout hundreds of thousands of scales with intricate microstructures and nano-structures that determine the wings' optical appearance, wetting characteristics, thermodynamic properties, and aerodynamic behavior. Although the functional characteristics of scales are well known and prove desirable in various applications, the dynamic processes and temporal coordination required to sculpt the scales' many structural features remain poorly understood. Current knowledge of scale growth is primarily gained from ex vivo studies of fixed scale cells at discrete time points; to fully understand scale formation, it is critical to characterize the time-dependent morphological changes throughout their development. Here, we report the continuous, in vivo, label-free imaging of growing scale cells of Vanessa cardui using speckle-correlation reflection phase microscopy. By capturing time-resolved volumetric tissue data together with nanoscale surface height information, we establish a morphological timeline of wing scale formation and gain quantitative insights into the underlying processes involved in scale cell patterning and growth. We identify early differences in the patterning of cover and ground scales on the young wing and quantify geometrical parameters of growing scale features, which suggest that surface growth is critical to structure formation. Our quantitative, time-resolved in vivo imaging of butterfly scale development provides the foundation for decoding the processes and biomechanical principles involved in the formation of functional structures in biological materials.

Structures and topological defects in pressure-driven lyotropic chromonic liquid crystals.

Lyotropic chromonic liquid crystals are water-based materials composed of self-assembled cylindrical aggregates. Their behavior under flow is poorly understood, and quantitatively resolving the optical retardance of the flowing liquid crystal has so far been limited by the imaging speed of current polarization-resolved imaging techniques. Here, we employ a single-shot quantitative polarization imaging method, termed polarized shearing interference microscopy, to quantify the spatial distribution and the dynamics of the structures emerging in nematic disodium cromoglycate solutions in a microfluidic channel. We show that pure-twist disclination loops nucleate in the bulk flow over a range of shear rates. These loops are elongated in the flow direction and exhibit a constant aspect ratio that is governed by the nonnegligible splay-bend anisotropy at the loop boundary. The size of the loops is set by the balance between nucleation forces and annihilation forces acting on the disclination. The fluctuations of the pure-twist disclination loops reflect the tumbling character of nematic disodium cromoglycate. Our study, including experiment, simulation, and scaling analysis, provides a comprehensive understanding of the structure and dynamics of pressure-driven lyotropic chromonic liquid crystals and might open new routes for using these materials to control assembly and flow of biological systems or particles in microfluidic devices.

Stomach tissue classification using autofluorescence spectroscopy and machine learning.

BACKGROUND AND OBJECTIVES: Determination of stomach tumor location and invasion depth requires delineation of gastric histological structure, which has hitherto been widely accomplished by histochemical staining. In recent years, alternative histochemical evaluation methods have been pursued to accelerate intraoperative diagnosis, often by bypassing the time-consuming step of dyeing. Owing to strong endogenous signals from coenzymes, metabolites, and proteins, autofluorescence spectroscopy is a favorable candidate technique to achieve this aim. MATERIALS AND METHODS: We investigated stomach tissue slices and block specimens using a fast fluorescence imaging scanner. To obtain histological information from broad and structureless fluorescence spectra, we analyzed tens of thousands of spectra with multiple machine-learning algorithms and built a tissue classification model trained with dissected gastric tissues. RESULTS: A machine-learning-based spectro-histological model was built based on the autofluorescence spectra measured from stomach tissue samples with delineated and validated histological structures. The scores from a principal components analysis were employed as input features, and prediction accuracy was confirmed to be 92.0%, 90.1%, and 91.4% for mucosa, submucosa, and muscularis propria, respectively. We investigated the tissue samples in both sliced and block forms using a fast fluorescence imaging scanner. CONCLUSION: We successfully demonstrated differentiation of multiple tissue layers of well-defined specimens with the guidance of a histologist. Our spectro-histology classification model is applicable to histological prediction for both tissue blocks and slices, even though only sliced samples were trained.

Bendable long graded index lens microendoscopy.

Graded index (GRIN) lens endoscopy has broadly benefited biomedical microscopic imaging by enabling accessibility to sites not reachable by traditional benchtop microscopes. It is a long-held notion that GRIN lenses can only be used as rigid probes, which may limit their potential for certain applications. Here, we describe bendable and long-range GRIN microimaging probes for a variety of potential micro-endoscopic biomedical applications. Using a two-photon fluorescence imaging system, we have experimentally demonstrated the feasibility of three-dimensional imaging through a 500-µm-diameter and ∼11 cm long GRIN lens subject to a cantilever beam-like deflection with a minimum bend radius of ∼25 cm. Bend-induced perturbation to the field of view and resolution has also been investigated quantitatively. Our development alters the conventional notion of GRIN lenses and enables a range of innovative applications. For example, the demonstrated flexibility is highly desirable for implementation into current and emerging minimally invasive clinical procedures, including a pioneering microdevice for high-throughput cancer drug selection.

Dual modal spectroscopic tissue scanner for colorectal cancer diagnosis.

BACKGROUND: Margin status is an important prognostic factor for treating colorectal cancer. This study aimed to investigate the usefulness of a multimodal spectroscopic tissue scanner for real-time cancer diagnosis without tissue staining. PATIENTS AND METHODS: Diffuse reflectance spectra (DRS) and fluorescence spectra (FS) of < 1-mm-sized paired cancer and normal mucosa tissue were acquired using custom-built spectroscopic tissue scanners. For FS, we analyzed wavelengths and intensities at peaks and highest intensities near (± 1.25 nm) the known fluorescence spectral peaks of collagen (380 nm), reduced nicotinamide adenine dinucleotide (NADH, 460 nm), and flavin adenine dinucleotide (FAD, 550 nm). For DRS, we performed a similar analysis near the peaks of strong absorbers, oxyhemoglobin (oxyHb; 414 nm, 540 nm, and 576 nm) and deoxyhemoglobin (deoxyHb; 432 nm and 556 nm). Logistic regression analysis for these parameters was performed in the testing set. RESULTS: We acquired 17,735 spectra of cancer tissues and 9438 of normal tissues from 30 patients. Intensity peaks of representative normal spectra for FS and DRS were higher than those of representative cancer spectra. Logistic regression analysis showed wavelength and intensity at peaks, and the intensities of the peak wavelength of NADH, FAD, deoxyHb, and oxyHb had significant coefficients. The area under the receiver operating characteristic curve was 0.927. The scanner had 100%, 64.3%, and 85.3% sensitivity, specificity, and accuracy, respectively. CONCLUSIONS: The spectroscopic tissue scanner has high sensitivity and accuracy and provides real-time intraoperative resection margin assessments and should be further investigated as an alternative to frozen section.

Absorption by water increases fluorescence image contrast of biological tissue in the shortwave infrared.

Recent technology developments have expanded the wavelength window for biological fluorescence imaging into the shortwave infrared. We show here a mechanistic understanding of how drastic changes in fluorescence imaging contrast can arise from slight changes of imaging wavelength in the shortwave infrared. We demonstrate, in 3D tissue phantoms and in vivo in mice, that light absorption by water within biological tissue increases image contrast due to attenuation of background and highly scattered light. Wavelengths of strong tissue absorption have conventionally been avoided in fluorescence imaging to maximize photon penetration depth and photon collection, yet we demonstrate that imaging at the peak absorbance of water (near 1,450 nm) results in the highest image contrast in the shortwave infrared. Furthermore, we show, through microscopy of highly labeled ex vivo biological tissue, that the contrast improvement from water absorption enables resolution of deeper structures, resulting in a higher imaging penetration depth. We then illustrate these findings in a theoretical model. Our results suggest that the wavelength-dependent absorptivity of water is the dominant optical property contributing to image contrast, and is therefore crucial for determining the optimal imaging window in the infrared.

Spectrochemical Probing of MicroRNA Duplex Using Spontaneous Raman Spectroscopy for Biosensing Applications.

MicroRNAs are emerging as both diagnostic and therapeutic targets in different human pathologies. An accurate understanding of the structural dependency of microRNAs for their biological functions is essential for designing synthetic oligos with various base and linkage modifications that can transform into highly sensitive diagnostic devices and therapeutic molecules. In this proof-of-principle study, we have utilized label-free spontaneous Raman spectroscopy to understand the structural differences in sense and antisense microRNA-21 by hybridizing them with complementary RNA and DNA oligos. Overall, the results suggest that the changes in the Raman band at 785 cm-1 originating from the phosphodiester bond of the nucleic acid backbone, linking 5' phosphate of the nucleic acid with 3' OH of the other nucleotide, can serve as a marker to identify these structural variations. Our results support the application of Raman spectroscopy in discerning intramolecular (ssRNA and ssDNA) and intermolecular (RNA-RNA, RNA-DNA, and DNA-DNA hybrids) interactions of nucleic acids. This is potentially useful for developing biosensors to quantify microRNAs in clinical samples and to design therapeutic microRNAs with robust functionality.

Analysis of subcutaneous swine fat via deep Raman spectroscopy using a fiber-optic probe.

Since the fat content of pork is a deciding factor in meat quality grading, the use of a noninvasive subcutaneous probe for real-time in situ monitoring of the fat components is of importance to vendors and other interested parties. In this work, we developed a spectroscopic method using a fiber-optic probe for subcutaneous fat analysis that utilizes spatially offset Raman spectroscopy (SORS). Here, normalized Raman spectra were acquired as a function of spatial offset, and the relative composition of fat-to-skin was determined. We found that the Raman intensity ratio varied disproportionately depending on the fat content and that the variations of the slope were correlated to the thickness of the fat layer. Furthermore, ordinary least square (OLS) regression using two components indicated that the depth-resolved SORS spectra reflected the relative thickness of the fat layer. We concluded that the local distribution of subcutaneous fat could be measured noninvasively using a pair of fiber-optic probes.

Quantification of labile heme in live malaria parasites using a genetically encoded biosensor.

Heme is ubiquitous, yet relatively little is known about the maintenance of labile pools of this cofactor, which likely ensures its timely bioavailability for proper cellular function. Quantitative analysis of labile heme is of fundamental importance to understanding how nature preserves access to the diverse chemistry heme enables, while minimizing cellular damage caused by its redox activity. Here, we have developed and characterized a protein-based sensor that undergoes fluorescence quenching upon heme binding. By genetically encoding this sensor in the human malarial parasite, Plasmodium falciparum, we have quantified cytosolic labile heme levels in intact, blood-stage parasites. Our findings indicate that a labile heme pool (∼1.6 µM) is stably maintained throughout parasite development within red blood cells, even during a period coincident with extensive hemoglobin degradation by the parasite. We also find that the heme-binding antimalarial drug chloroquine specifically increases labile cytosolic heme, indicative of dysregulation of this homeostatic pool that may be a relevant component of the antimalarial activity of this compound class. We propose that use of this technology under various environmental perturbations in P. falciparum can yield quantitative insights into fundamental heme biology.

Eleven quick tips for running an interdisciplinary short course for new graduate students.

Quantitative reasoning and techniques are increasingly ubiquitous across the life sciences. However, new graduate researchers with a biology background are often not equipped with the skills that are required to utilize such techniques correctly and efficiently. In parallel, there are increasing numbers of engineers, mathematicians, and physical scientists interested in studying problems in biology with only basic knowledge of this field. Students from such varied backgrounds can struggle to engage proactively together to tackle problems in biology. There is therefore a need to establish bridges between those disciplines. It is our proposal that the beginning of graduate school is the appropriate time to initiate those bridges through an interdisciplinary short course. We have instigated an intensive 10-day course that brought together new graduate students in the life sciences from across departments within the National University of Singapore. The course aimed at introducing biological problems as well as some of the quantitative approaches commonly used when tackling those problems. We have run the course for three years with over 100 students attending. Building on this experience, we share 11 quick tips on how to run such an effective, interdisciplinary short course for new graduate students in the biosciences.

Single-shot dual-wavelength interferometric microscopy.

Interferometric microscopy (IM) can provide complex field information of the biological samples with high spatial and temporal resolution with virtually no photodamage. Measuring wavelength-dependent information in particular has a wide range of applications from cell and tissue refractometry to the cellular biophysical measurements. IM measurements at multiple wavelengths are typically associated with a loss in temporal resolution, field of view, stability, sensitivity, and may involve using expensive equipment such as tunable filters or spatial light modulators. Here, we present a novel and simple design for an interferometric microscope that provides single-shot off-axis interferometric measurements at two wavelengths by encoding the two spectral images at two orthogonal spatial frequencies that allows clean separation of information in the Fourier space with no resolution loss. We demonstrated accurate simultaneous quantification of polystyrene bead refractive indices at two wavelengths.

Correction to: Evaluation of accuracy dependence of Raman spectroscopic models on the ratio of calibration and validation points for non-invasive glucose sensing.

The authors would like to bring to the reader's attention that the Clarke error grid plot presented in Fig. 3 was generated using codes adapted from following reference.

Increasing the penetration depth of temporal focusing multiphoton microscopy for neurobiological applications.

The first ever demonstration of temporal focusing with short wave infrared (SWIR) excitation and emission is demonstrated, achieving a penetration depth of 500 µm in brain tissue. This is substantially deeper than the highest previously-reported values for temporal focusing imaging in brain tissue, and demonstrates the value of these optimized wavelengths for neurobiological applications.

Cellular normoxic biophysical markers of hydroxyurea treatment in sickle cell disease.

Hydroxyurea (HU) has been used clinically to reduce the frequency of painful crisis and the need for blood transfusion in sickle cell disease (SCD) patients. However, the mechanisms underlying such beneficial effects of HU treatment are still not fully understood. Studies have indicated a weak correlation between clinical outcome and molecular markers, and the scientific quest to develop companion biophysical markers have mostly targeted studies of blood properties under hypoxia. Using a common-path interferometric technique, we measure biomechanical and morphological properties of individual red blood cells in SCD patients as a function of cell density, and investigate the correlation of these biophysical properties with drug intake as well as other clinically measured parameters. Our results show that patient-specific HU effects on the cellular biophysical properties are detectable at normoxia, and that these properties are strongly correlated with the clinically measured mean cellular volume rather than fetal hemoglobin level.

Three-dimensional super-resolution high-throughput imaging by structured illumination STED microscopy.

Stimulated emission depletion (STED) microscopy is able to image fluorescence labeled samples with nanometer scale resolution. STED microscopy is typically a point-scanning method, limited by the high intensity requirement of the depletion beam. With the development of high peak power lasers, two dimensional parallel STED microscopy has been developed. Here, we develop the theoretical basis for extending STED microscopy to three dimensional imaging in parallel. This method uses structured illumination (SI) to generates a three dimensional depletion pattern. Compared to the two dimensional parallel STED microscopy, the 3D SI-STED microscopy generates intensity modulation along the light propagation direction without requiring higher laser power. This method not only achieves axial super-resolution of STED microscopy but also greatly reduces photobleaching and photodamage for 3D volumetric imaging.

Dynamic spatial filtering using a digital micromirror device for high-speed optical diffraction tomography.

Optical diffraction tomography (ODT) is an emerging microscopy technique for three-dimensional (3D) refractive index (RI) mapping of transparent specimens. Recently, the digital micromirror device (DMD) based scheme for angle-controlled plane wave illumination has been proposed to improve the imaging speed and stability of ODT. However, undesired diffraction noise always exists in the reported DMD-based illumination scheme, which leads to a limited contrast ratio of the measurement fringe and hence inaccurate RI mapping. Here we present a novel spatial filtering method, based on a second DMD, to dynamically remove the diffraction noise. The reported results illustrate significantly enhanced image quality of the obtained interferograms and the subsequently derived phase maps. And moreover, with this method, we demonstrate mapping of 3D RI distribution of polystyrene beads as well as biological cells with high accuracy. Importantly, with the proper hardware configuration, our method does not compromise the 3D imaging speed advantage promised by the DMD-based illumination scheme. Specifically, we have been able to successfully obtain interferograms at over 1 kHz speed, which is critical for potential high-throughput label-free 3D image cytometry applications.

Reference-free polarization-sensitive quantitative phase imaging using single-point optical phase conjugation.

We propose and experimentally demonstrate a method of polarization-sensitive quantitative phase imaging using two photodetectors and a digital micromirror device. Instead of recording wide-field interference patterns, finding the modulation patterns maximizing focused intensities in terms of the polarization states enables polarization-dependent quantitative phase imaging without the need for a reference beam and an image sensor. The feasibility of the present method is experimentally validated by reconstructing Jones matrices of several samples including a polystyrene microsphere, a maize starch granule, and a mouse retinal nerve fiber layer. Since the present method is simple and sufficiently general, we expect that it may offer solutions for quantitative phase imaging of birefringent materials.

In vivo detection of drug-induced apoptosis in tumors using Raman spectroscopy.

We describe a label-free approach based on Raman spectroscopy, to study drug-induced apoptosis in vivo. Spectral-shifts at wavenumbers associated with DNA, proteins, lipids, and collagen have been identified on breast and melanoma tumor tissues. These findings may enable a new analytical method for rapid readout of drug-therapy with miniaturized probes.

Evaluation of accuracy dependence of Raman spectroscopic models on the ratio of calibration and validation points for non-invasive glucose sensing.

Optical monitoring of blood glucose levels for non-invasive diagnosis is a growing area of research. Recent efforts in this direction have been inclined towards reducing the requirement of calibration framework. Here, we are presenting a systematic investigation on the influence of variation in the ratio of calibration and validation points on the prospective predictive accuracy of spectral models. A fiber-optic probe coupled Raman system has been employed for transcutaneous measurements. Limit of agreement analysis between serum and partial least square regression predicted spectroscopic glucose values has been performed for accurate comparison. Findings are suggestive of strong predictive accuracy of spectroscopic models without requiring substantive calibration measurements. Graphical abstract.